African Journal of Biotechnology Vol. 10(7), pp. 1194-1199, 14 February, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.1432 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Rapid micropropagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl. (a valuable medicinal plant) from shoot bud explants Nor Azma Yusuf 1,2 *, M. M. Suffian Annuar 1 and Norzulaani Khalid 1 1Biotechnology and Bioproduct Research Cluster, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Faculty of Plantation and Agro biotech, Faculty of Applied Sciences, University Technology MARA (Uitm) 40450 Shah Alam, Selangor, Malaysia. Accepted 7 December, 2010 A successful protocol was developed for mass propagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl., an important medicinal plant. Numerous shoots were induced from young shoot bud of B. rotunda mature rhizome on Murashige and Skoog (1962) medium supplemented with 30.0 g/l sucrose, 2.0 g/l gelrite, different concentrations of 6-benzylaminopurine (BAP) and α-naphtaleneacetic acid (NAA). Plant medium supplemented with different concentrations of BAP alone or with NAA produced varying degree of multiple shoots. A supplementation of 2.0 mg/l BAP and 0.5 mg/l NAA gave the best result. Ninety percent of the explants induced multiple shoots within 10 to 14 days of inoculation with five maximum numbers of shoots per explant. The numbers of multiple shoots was low during initial subculture but increased after third subculture and were slightly decreased after fourth subculture. Rooting was spontaneous in almost all the treatments after 10 to 14 days of culture. Micropropagated plantlets were successfully acclimatized. Key words: Boesenbergia rotunda , micropropagation, medicinal plant, Zingiberaceae. INTRODUCTION Boesenbergia rotunda (L) Mansf. Kulturpfl. (Larsen, 1989; Murakami et al., 1993; Tuchnida et al., 2002; 1996), is a perennial herbaceous plant that belongs to the Fahey and Stephenson, 2002; Tewtrakul et al., 2003). In family Zingiberaceae and is believed to have originated a recent study, 4-hydroxypanduratin A and panduratin A, from India and South-East Asia region. The pharma- isolated from the rhizome of B. rotunda were found to cological importance of this plant is mainly due to the show high inhibitory activity towards dengue-2 virus pro- presence of flavanoids, essential oil and chalcones tease at 120 ppm (Tan et al., 2006). (Jaipetch et al., 1982 ; Pandji et al., 1993; Trakoontivakorn B. rotunda is traditionally propagated by vegetative et al., 2001). Moreover, rhizomes contain a potential techniques using a rhizome segment which is lengthy for antipyretic, analgesic, anti-mutagenic, anti-inflammatory, large-scale multiplication. Moreover, many of the Zingibe- antioxidant enzymes and anti-human immunodeficiency raceae species are susceptible to rhizome and soft rot virus 1 (anti-HIV-1) protease inhibition (Pathong et al., diseases, leaf spot and easily infected with pathogens such as Coleotrichum species (Balachandran et al ., 1990; Chan and Thong, 2004). A rapid multiplication method is required to supply sufficient disease-free plant material for large scale cultivation using in vitro propa- *Corresponding author. E-mail: [email protected] or gation. In vitro propagation of Zingiberaceae has already [email protected]. Tel: +603 79674380. Fax: +603 79674178. been reported, for example Alpinia galanga (Inden and Abbreviations: BAP, N6-Benzylaminopurine; NAA, α- Asahir , 1988) and Zingiber officinale (Hosoki and Sagawa, naphthalene acetic acid; MS, Murashige and Skoog; HIV, 1977; Pillai and Kumar, 1982; Inden and Asahira, 1988; human immunodeficiency virus. Bhagyalakshmi and Singh, 1988; Ikeda and Tanabe Yusuf et al. 1195 1989). Several investigators reported micropropagation of Acclimatization ginger either from buds (Nadgauda et al., 1980) or shoot In vitro rooted plantlets of B. rotunda (Figure 1E) were removed tips (Inden and Asahira, 1988), meristems (Bhagyalakshmi from the solid MS medium and the roots were washed under and Singh, 1988), or even from callus (Malamug et al., running tap water to remove residual agar. Each plantlet was then 1991; Nirmal et al., 1992). In vitro clonal multiplication of planted into pots containing mixture of organic soil and sand (1:1) Curcuma species through rhizome buds also has been without direct sunlight. The percentage of surviving plantlets was reported (Nadgauda et al., 1978; Balachandran et al., recorded after four weeks of transfer. 1990; Barthakur and Bordoloi, 1992; Salvi et al., 2002; Loc et al., 2005, Yusuf et al., 2007; Naz et al., 2009). How- ever , to date, there are no reports on the rapid micropropa- RESULTS AND DISCUSSION gation of B. rotunda , which will be discussed in this paper. Initiation of shoot buds MATERIALS AND METHODS Establishment of contamination free cultures was a major task due to the fact that the explants originated from Establishment of aseptic explants underground rhizomes (Hosoki and Sagawa, 1977). In this study, rhizomes were initially sprouted on soil free Mature rhizomes of B. rotunda were purchased from herbal supplier condition until shoot buds appearred. These shoot buds at a local Market in Kuala Lumpur, Malaysia. The rhizomes were cleaned and rinsed. The cleaned rhizomes were then placed in an were excised from rhizomes and were used as explants. open container to allow shoots to sprout two to four cm in length. The buds from the rhizomes of B. rotunda that were The sprouting buds were collected and washed with 20% (v/v) surface sterilized with mercury chloride solution and com- clorox for 15 min. Under aseptic conditions, shoots were surface mercial bleach (Clorox ) solution for 15 min could sterilized with 0.5% (w/v) aqueous solution of mercuric chloride establish more than 70% aseptic and surviving explants (HgCl 2) solution for five minutes and followed by three rinses in sterile distilled water. The shoot buds had their external leaves and remained free of contamination after four (4) weeks removed and trimmed down until the size ranged from 0.5 to 0.8 in MS medium. Chan and Thong (2004) also reported mm and used as explants (Figure 1A to C). They were then that the use of HgCl 2 with two-stage surface sterilization inoculated into 350 ml glass jar containing MS (Murashige and using Clorox solution was extremely efficient for Skoog, 1962) medium supplemented with 30.0 g/l sucrose and 2.0 establishing aseptic buds of other Zingiberaceae species. g/l gelrite for 30 days. The pH of the medium was adjusted to 5.7 prior to autoclaving at 121°C for 21 min. The cultures were Similar sterilization process was also used to establish maintained in a culture room with 16/8 h photoperiod (light/dark) at the aseptic buds of Cymbopogon nardus (Chan et al., 25 ± 2°C. After 30 days, the aseptic buds of this species were 2005). Once the contamination free cultures of the shoot transferred onto MS medium supplemented with 3.0 mg/l BAP and buds were established, they were easily maintained by 0.5 mg/l NAA, the optimum medium was formulated for other sub culturing on fresh medium. Zingiberaceae species by Yusuf et al. (2007) for shoot multiplication for four (4) weeks (Figure 1D). The multiple shoots produced were separated and transferred into MS medium devoid of plant growth regulators for four (4) weeks and were used for all subsequent Shoots growth and multiplication experiments. The aseptic shoots of B. rotunda cultured on MS medium supplemented with different concentrations of BAP alone Induction of multiple shoots (0.0 to 5.0 mg/l) or in combinations with NAA (0.0 to 2.0 mg/l) resumed their growth, produced shoots and roots, Isolated shoot buds were inoculated onto MS (Murashige and Skoog, 1962) medium containing 30.0 g/l sucrose and 2.0 g/l gelrite simultaneously (Table 1). The simultaneous production of as solidifying agent supplemented with cytokinins (6-benzyla- shoots and roots were also reported in other Zingibe- minopurine (BAP)) ranging from 0.0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l raceae species (Balachandran et al., 1990; Chan and and auxin (α-naphthaleneacetic acid (NAA)) ranging from 0.0, 0.5, Thong, 2004; Bharalee et al. , 2005). The growth of the 1.0 and 2.0 mg/l, for shoot multiplication. A single shoot was shoots and subsequent multiplication could not be cultured into each 350 ml glass jar and 10 experimental units were achieved in medium without growth regulators. After 4 to used for each medium combination. 6 weeks, about 90% of the shoots of B. rotunda that were cultured on MS medium supplemented with 0.5 to 3 mg/l Scoring of data of BAP and 0.5 mg/l NAA showed different levels of development with high micropropagation frequency All cultures were examined periodically, and the morphological (induced 4 to 5 multiple shoots, Table 1) and variable changes were recorded on the basis of visual observations and number of leaves with 10 to 15 roots hairs including were made after one to four weeks of culture for four consecutive secondary roots per explants (data not shown). The subcultures. There were 10 cultures per treatment for shoot multi- maximum number of multiple shoots (5) was obtained in plication and subculturing was carried out at an interval of four weeks. The effect of different treatments and subcultures were the medium containing 2.0 mg/l of BAP and 0.5 mg/l NAA quantified on the basis of percentage of cultures showing response four (4) weeks after culture initiation (Figure 2). However, for multiple shoots formation. the buds cultured on MS medium containing higher 1196 Afr. J. Biotechnol. Figure 1. In vitro regeneration and plant establishment of B. rotunda . (A) Shoots sprouted from mature rhizomes of B. rotunda . Bar represents 25 mm. (B) Sprouted buds (30 to 50 mm) collected as explants.